A panel of CYP6Z genes drives broad-spectrum cross-resistance to public health insecticides in the major malaria vector An. gambiae s.s.

The spread of multiple and intense insecticide resistance in major African malaria vectors is jeopardising control efforts. Mitigating this threat requires deciphering its underlying molecular mechanisms. Here, by integrating transcriptomic profiling, analysis of genetic diversity and functional genomics approaches, we established the role of the CYP6Z genes in conferring multiple- and cross-resistance to insecticides. Investigation of CYP6Z gene expression and genetic diversity analyses indicate that resistance is primarily associated with transcriptional upregulation rather than fixed coding mutations since no predominant haplotype was selected. Structural characterisation reveals a flexible, promiscuous active site in CYP6Z enzymes, enabling binding of multiple insecticide classes. In vitro functional validation confirms that recombinant CYP6Z3 efficiently metabolizes deltamethrin (percentage depletion of 59%), permethrin (52%), -cypermethrin (37%), pirimiphos-methyl (36%), fenitrothion (25%), propoxur (25%), and possibly bendiocarb (17%). RNAi-mediated knockdown of CYP6Z genes in field-collected An. gambiae s.s. restores susceptibility to several insecticides including clothianidin: dsCYP6Z1 (mortality = 50.29%; p < 0.01), dsCYP6Z2 (47.28%; p < 0.01) and dsCYP6Z3 (39.12%; p < 0.05) compared to the control (mortality = 27.67%). Furthermore, transgenic expression in Drosophila melanogaster flies revealed that expression of CYP6Z genes alone confer cross-resistance to pyrethroids, organophosphates, carbamates and neonicotinoids, but increases susceptibility to the pro-insecticide chlorfenapyr: CYP6Z1 (mortality = 100%; p < 0.01), CYP6Z3 (98.18%; p < 0.01), and CYP6Z4 (98.01%; p < 0.01) vs. control (88.03%). This study establishes An. gambiae CYP6Z genes operate in concert to drive a broad-spectrum metabolic resistance, towards several classes of public health insecticides, while in contrast bioactivating chlorfenapyr to its more insecticidally active metabolite.